The role of high mobility group box 1 in the signaling pathways of mouse intestinal ischemia-reperfusion injury / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the role of high mobility group box 1 (HMGB1) in the signaling pathway of mouse intestinal ischemia-reperfusion injury.</p><p><b>METHODS</b>Twenty-four Specific Pathogen free male C57BL / 6 mice were randomly divided into three groups (n = 8) : the sham operation group (sham), the control group(control) and the HMGB1 antibody group (anti-HMGB1). The vehicle alone or anti-HMGB1 antibody(1 mg/kg, 0. 025%) was injected respectively via the caudal vein 30 min prior to ischemia in the control group or the anti-HMGB1 group. All mice were anesthetized,opened abdominal wall and exposed arteria mesenterica superior. The control group and the anti-HMGBl group underwent 60 min of mesenteric ischemia and 60 min of reperfusion and the sham group were merely opened abdominal wall for 120 min without ischemia-reperfusion. The levels of NF-κB p65, IL-6 and TNF-α in plasma and the activity of MPO in lung and liver and the morphological changes of lung and intestinal tissue were measured. The mRNA levels of HMGB1 and NF-κB were evaluated using real-time quantitative PCR and the protein levels of HMGB1 and NF-KB were evaluated using Western blot. The experimental data was analyzed using one-way analysis of variance.</p><p><b>RESULTS</b>The levels of IL-6, TNF-α and NF-κB p65 in plasma was significantly higher in the control group and the anti-HMGB1 group compared with the sham group (the sham group vs. the control group vs. the anti-HMGB1 group, NF-κB p65, 104. 64 ± 11. 89: 228. 53 ± 24. 85: 145. 00 ± 33. 63, F = 38. 036, P <0. 05; IL-6,50. 02 ± 6. 33:104. 91 ± 31. 18:62. 28 ± 6. 73, F = 49. 763, P < 0. 05; TNF-α, 43. 79 ± 4. 18: 70. 81 ± 6. 97: 52. 76 ± 5. 71, F = 34. 571, P < 0. 05). The increasing degree in the anti- HMGB1 group was significantly reduced compared with the control group (P <0. 05). The activity of MPO of liver and lung in the control group and the anti-HMGB1 group was significantly higher than those in the sham group (P <0. 05). Compared with the sham group, the degree of tissue injury in jejunum, ileum and lung was serious in the control group, and that in the anti-HMGB1 group was significantly lower than the control group. The expression of HMGB1 mRNA and NF-κB mRNA in the lung and the ileum in the sham group and the control group were all higher than the sham group (HMGB1 mRNA in lung: sham group 1. 04 ± 0. 19 vs. control group 2. 25 ± 0. 18 vs. anti-HMGB1 group 1. 89 0. 18, F = 66. 203, P < 0. 05; in ileum: 1. 14 ± 0. 54 vs. 6. 26 ± 0. 60 vs. 4. 93 0. 55, F = 133. 427, P < 0. 05; NF-κB mRNA in lung: 1. 03 ± 0. 21 vs. 2. 04 ± 0. 29 vs. 1. 42 ± 0. 23, F =26. 229, P < 0. 05; ileum: 1. 03 ± 0. 23 vs. 3. 71 ± 0. 53 vs. 2. 23 ± 0. 55, F = 50. 477, P <0. 05). Subjected to intestinal ischemia-reperfusion injury, the protein expression of HMGB1 and NF-κB in the lung, jejunum and ileum in the control group and the anti-HMGB1 group increased compared with the sham group(P <0. 05), but that was significantly lower in the anti-HMGB1 group than the control group (P <0. 05).</p><p><b>CONCLUSION</b>The administration of anti-HMGB1 antibodies may reduce the damage caused by ischemia-reperfusion effectively.</p>

The effects of lymph after intestinal ischemia/reperfusion on the inflammatory factors and Toll-like receptor 4 ligand high mobility group box-1 in Toll-like receptor 4 deficient mice / 中华临床营养杂志

Objective To investigate the effects of lymph from ischemic/reperfused intestine on the inflammatory factors and Toll-like receptor 4 (TLR4) ligand high mobility group box-1 (HMGB1) in TLR4 deficient (TLR4-/-) mice.Methods A total of 20 SD rats weighing (300 ±20) g were randomly assigned into two groups:lymph drainage group (group N,lymph drainage for 180 minutes without other treatment) and intestinal ischemia/reperfusion group (group I/R,draining the lymph for 180 minutes while clipping the superiormesenteric artery for 60 minutes followed by 120-minute reperfusion).Thirty-two TLR4-/-mice and thirty-two C57BL/6 wild type (WT) mice were each divided into 4 sub-groups (n =8),injected with different fluids through the caudal vein:group N with normal lymph;group I/R with I/R lymph;group Edt with endotoxin;group HMGB1 with HMGB1 protein.The mice were sacrificed 180 minutes after the injection for sample collection.Results The levels of endotoxin and HMGB1 in the lymph drainage of the group I/R rats were significantly higher than that of the group N rats [(0.034 ± 0.050) Eu/ml vs.(0.017 ± 0.023) Eu/ml,P =0.033;(4.293 ± 0.883) ng/ml vs.(0.509 ± 0.128) ng/ml,P =0.006].In the mice injected with HMGB1,the mucosa thickness and villus height in the ileum of the WT mice were significantly lower than that of the TLR4-/-mice [(335.8±43.2) μmvs.(602.1±37.5) μm,P=0.000;(273.0±31.7) μm vs.(404.5 ± 18.6) μm,P =0.000];in both WT and TLR4-/-mice injected with the I/R lymph drainage,the mucosa thickness and virus height were decreased,but the decrements were significantly lower in TLR4-/-mice;there were no statistically significant differences in the levels of interleukin-6 (IL-6),tumor necrosis factor-α (TNF-α),endotoxin,and HMGB1 between the TLR4-/-and the WT mice injected with normal lymph or endotoxin.In the mice injected with I/R lymph drainage,the levels of inflammatory factors in the TLR4-/-mice were significantly lower than those in the WT mice [TNF-α:(28.637 ±5.166) pg/ml vs.(41.917 ±8.175) pg/ml,P=0.000;IL-6:(60.900 ±24.729) pg/ml vs.(110.265 ±28.545) pg/ml,P =0.000].In the mice injected with HMGB1,the levels of inflammatory factors in the TLR4-/-mice were significantly decreased compared with those in the WT mice [TNF-α:(20.865 ± 6.464) pg/ml vs.(31.059 ± 6.204) pg/ml,P=0.004;IL-6:(36.268 ±8.977) pg/ml vs.(76.677 ± 14.099) pg/ml,P=0.000].Conclusions The concentrations of endotoxin and HMGB1 are significantly increased during intestinal I/R in rats.After injection of I/R lymph drainage,endotoxin,and HMGB1,the levels of inflammatory factors and HMGB1 in the mice injected with I/R lymph drainage are significantly higher than those in the mice injected with normal lymph;the levels of inflammatory factors and local damage of intestinal mucosa are significantly reduced in the TLR4-/-mice than in the WT mice.The gut-lymph pathway may play a key role in the intestinal I/R injury.

Correlations among different measurement indicators of intestinal barrier / 中华临床营养杂志

Objective To study several measuring methods of the intestinal mucosa barrier and evaluate the correlation between different methods for determining the intestinal damage.Methods Sixteen specific pathogen free (SPF) male Sprague-Dawley rats were randomly divided into two groups:the Control group (n =8) and the ischemia/reperfusion (I/R) group (n =8).After adaptive feeding for 5 days,I/R group was put into ischemia model for 60 min and the Control group was merely opened on its abdominal wall but without ischemia for 60 min.After having been fed for another day,all rats were killed and specimens were collected.The plasma diamine oxidase (DAO),D-lactate (D-LAC),endotoxin,and glutamine (Gln) levels were detected,and the intestinal mucosal morphology was observed.The intestinal permeability (L/M) was detected 1 day before and after the surgery.Results The plasma DAO,D-LAC,and endotoxin levels were significantly higher in I/R group compared with the Control group (DAO:(0.498 ±0.032) vs (0.247 ±0.051) U/ml,t=-11.790,P=0.000; D-LAC:(5.47±1.55) vs (3.83±0.63) mg/L,t=-2.757,P=0.022; endotoxin:(0.0395±0.002 8) vs (0.025 6 ±0.004 5) EU/ml,t =-7.377,P =0.000).The plasma Gln concentration was significantly lower than that in the Control group [(646.12 ± 34.75) vs (839.13 ± 163.76) μmol/L,t =3.261,P =0.012).The L/M value on the 1 st postoperative day was significantly higher than that in the I/R group [(3.63 ±2.09) vs (1.22 ±0.66),t =-3.118,P =0.013)].The jejunum mucosal thickness,jejunum villus height,ileal mucosal thickness,and ileal villus height were significantly lower in I/R group compared with the Control group after operation [(329.80 ±64.68) vs (512.82 ±38.41) μm,t=6.881,P=0.000; (253.06±69.33) vs (386.79±56.39) μm,t=4.232,P=0.001; (205.89± 18.71) vs (335.29±27.71) μm,t=10.945,P=0.000; (135.61 ±22.30) vs (253.18±31.02) μm,t =8.705,P =0.000].After intestinal ischemia/reperfusion,DAO,D-LAC,endotoxin and L/M were all increased and positively correlated with each other.The plasma concentration of Gln and the morphological changes of jejunum and ileum were negatively correlated with DAO,D-LAC,endotoxin,and L/M,respectively.Conclusions After intestinal ischemia/rcperfusion,the levels of all examination indicators obviously change and correlate with each other.The DAO,D-LAC,endotoxin,and L/M are positively related to each other and negatively correlated with the intestinal barrier function.Gln is positively correlated with small intestinal mucosal morphology and negatively correlated with others,respectively.

Impact of intestinal ischemia/reperfusion on free amino acid levels in plasma / 中华临床营养杂志

Objective To investigate intestinal mucosal injury and the change of free amino acid levels in plasma with intestinal ischemia/reperfusion.Methods Twenty-four Sprague-Dawley (SD) male rats (SPF grade) were randomly divided into 3 groups with 8 rats in each group:Blank group,Sham group and ischemia/reperfusion (I/R) group.The rats in I/R group were subjected to 60 min ischemia by clamping the superior mesenteric artery (SMA),followed by 120 min repeffusion.All rats were sacrificed with blood withdraw through inferior vena cava.The plasma was precipitated with Sulfosalicylic acid and the supernatant free amino acid levels were measured and the intestinal mucosal thickness and villus length were also assayed.Results In the I/R group the total free amino acids,essential amino acids (EAA),glutamine and branched-chain amino acids (BCCA) were remarkably lower [the total free amino acids:I/R vs Blank vs Sham:(4585.1 326.1) vs (5661.5 ±581.9) vs (5337.9±998.7) μmol/L (F=5.075,P=0.016); EAA:I/Rvs Blank vs Sham:(1401.3 ±183.4) vs (2147.6 ± 265.1) vs (1796.2 ± 440.8) μmol/L (F =1 1.216,P =0.000) ; glutamine:I/R vs Blank vs Sham:(646.1 ± 34.7) vs (895.7 ± 258.8) vs (839.1 ± 163.7) μmol/L (F =4.326,P =0.027) ; BCCA:I/R vs Blank vs Sham:(507.8 ± 119.0) vs (912.2 ± 165.8) vs (671.9 ± 79.8) μmol/L (F =10.662,P =0.001)]and the jejunum and ileum mucosal thickness and villus height were decreased compared to Blank and Sham groups [jejunum mucosal thickness:I/R vs Blank vs Sham:(401.50 ± 117.79) vs (529.22 ±54.73) vs (499.54 ±64.48) μm (F=31.869,P =0.000) ; jejunum villus height:I/R vs Blank vs Sham:(271.37 ± 84.29) vs (365.26 ± 46.98) vs (349.67 ± 56.11) μm (F =30.472,P =0.000) ; ileum mucosal thickness:I/R vs Blank vs Sham:(254.20 ± 43.56) vs (324.70 ± 30.56) vs (298.26 ± 58.46) μm (F =30.442,P =0.000) ; ileum villus height:I/R vs Blank vs Sham:(169.37 ± 37.25) vs (221.62 ± 37.26) vs (193.25 ± 38.39) μm (F =24.145,P =0.000)],and The EAA and BCAA in the I/R group were lower than the Sham group (respectively,P ＜0.05).There was no significant difference in aromatic amino acids (AAA) among the three groups [I/R vs Blank vs Sham:(273.2 ± 37.4) vs (296.8 ± 55.6) vs (281.9 ± 7.3) μmol/L (F =0.578,P =0.570)].The ratio BCAA/AAA in the Sham and I/R groups were significantly lower than the Blank group [(I/R vs Blank vs Sham:(2.4 ±0.6) vs.(1.9 ±0.4) vs (3.1 ±0.7) (F =5.215,P =0.014)],while the I/R group was decreased slightly compared to the Sham group,but the difference was not significant (P ＞ 0.05).The ethanolamine phosphate,taurine,citrulline,cystine,phosphoserine levels were reduced in the Sham and I/R groups compared to the Blank group [ethanolamine phosphate:I/R vs Blank vs Sham:(11.4 ± 1.9) vs (14.3 ± 3.4) vs (10.1±1.7) μmol/L(F=5.897,P=0.009);taurine:I/R vs BlankvsSham:(341.1±36.3) vs(533.2±90.8) vs (439.2±105.4) μmol/L (F=10.702,P=0.001); citrulline:I/R vs Blank vs Sham:(57.7±3.2) vs (73.1 ±16.2) vs (58.1 ±3.8) μmol/L (F=6.360,P =0.007); cystine:I/R vs Blank vs Sham:(20.0 ± 3.6) vs (60.6 ± 24.6) vs (36.3 ± 5.8) μmol/L (F =15.344,P =0.000) ; phosphoserine:I/R vs BlankvsSham:(10.2±1.1) vs (15.8±5.4) vs (11.7 ±3.4) μmol/L (F=4.878,P=0.018)],and the taurine and cystine in I/R groups were significantly decreased than the Sham group (respectively,P ＜ 0.05).The ornithine and arginine were comparatively reduced in I/R in contrast to the Blank and Sham groups [ornithine:I/R vs Blank vs Sham:(81.5 ± 19.0) vs (125.5 ±42.3) vs (114.9 ± 19.5) μmol/L (F =4.961,P =0.017) ;arginine:I/R vs Blank vs Sham:(199.2 ± 8.0) vs (258.9 ± 14.6) vs (248.7 ± 38.4) μmol/L (F =13.940,P =0.000)].The tryptophan and glutamic acid concentrations were increased in the Sham and I/R groups [tryptophan:L/R vs Blank vs Sham:(125.9 ± 12.1) vs (103.1 ± 29.9) vs (128.9 ± 18.5) μmol/L (F =5.429,P =0.031) ; glutamic acid:I/R vs Blank vs Sham:(188.6 ± 29.8) vs (93.6 ± 29.4) vs (125.4 ± 43.8) μmol/L (F =15.241,P =0.000)] and it was lower in the Sham group than the I/R group (P ＜ 0.05).Conclusion Intestinal ischemia-reperfusion can cause intestinal mucosal injury and the change of free amino acid levels in plasma and intestinal barrier damage may be related to the decline glutamine concentration and the increase of protein catabolism.

Absorption and transport mechanism of small peptides and their physiological functions / 中华临床营养杂志

Small peptides is one of the main components in the final product of protein digestion in the gastrointestinal tract,which plays an important role in protein nutrition.Present studies show that small peptides in the intestine can be absorbed directly into the circulation,which is also the main form of protein absorption in vivo.However,the transporter system of small peptides is independent from that of amino acids.This paper elaborates on the absorption and transport system of small peptides,their advantages in enteral nutrition,and some small peptides with critical physiological functions.

Effects of ω-3 polyunsaturated fatty acids intervention on Toll-like receptor 4 and high mobility group box 1 with intestinal ischemia/reperfusion injury / 中华临床营养杂志

Objective To investigate the expressions of Toll-like receptor 4 (TLR4) and high mobility group box 1 (HMGB1) expression on distant tissue during the intestinal ischemia/reperfusion and the effects of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) intervention in rats.Methods Forty-eight Sprague-Dawley male rats,weighing (281.50 ± 22.68) g,were randomly divided into three groups (n =16) after gastrostomy:normal diet (N) group,enteral nutrition (EN) group and EN plus ω-3 PUFAs (PUFA) group.Each group was further divided into lymph drainage (I/R + D) and non-drainage (I/R) sub-groups (n =8 each) according to whether treated with intestinal lymph drainage.All the rats were subjected to 60 min ischemia by clamping the superior mesenteric artery,followed by 120 min reperfusion,while the rats in the I/R + D subgroups were treated with intestinal lymph drainage for 180 min at the same time.Results The interleukin-6 level in lymph in N (I/R + D) group was significantly higher than in the EN (I/R + D) and PUFA (I/R + D) groups (PUFA vs EN vs N:(154.57 ±69.30) ng/L vs (97.58 ±40.34) ng/L vs (85.35 ±23.93) ng/L,P =0.021).Besides,the serum level of HMGB1 in PUFA (I/R + D) group was significantly lower compared to the other 5 groups [PUFA (I/R) vs EN (I/R) vs N (I/R) vs PUFA (I/R + D) vs EN (I/R + D) vs N (I/R + D):(2.95 ± 1.17) μg/L vs (3.86 ±0.99) μg/L vs (4.45 ± 1.73) μg/L vs (1.71 ±1.41) μg/Lvs (2.11±0.56) μg/Lvs (3.13 ±0.79) μg/L,P=0.000],and it also decreased in the PUFA (I/R) and EN (I/R) groups than the N (I/R) group (respectively,P ＜ 0.05).Furthermore,the serum endotoxin level in PUFA (I/R) group was significantly lower compared to the N (I/R) and EN (I/ R) groups[PUFA(I/R) vsPUFA (I/R+D) vsEN (I/R) vs N (I/R):(0.020±0.004) EU/mlvs (0.028 ±0.006) EU/ml vs (0.028 ±0.005) EU/ml vs (0.018 ±0.006) EU/ml,P=0.014].Together the serum tumor necrosis factor-α level in both PUFA (I/R) and PUFA (I/R + D) groups were significantly lower than theEN (I/R),N (I/R) and N (I/R+D) groups [PUFA (I/R+D) vs PUFA (I/R) vs EN (I/R) vsN (I/R) vs N (I/R+D):(12.03 ±6.57) ng/L vs (14.32 ±6.11) ng/Lvs (23.27 ±15.60)ng/L vs (27.42 ± 10.37) ng/L vs (26.87 ± 5.30) ng/L,P =0.013].The jejunum and ileum mucosa in all the I/R groups showed swelling and atrophy and appeared fragile,while the PUFA groups showed less yellow staining and injury than the other two groups (P ＜ 0.05,respectively).In addition,the expressions of TLR4 mRNA in jejunum,ileum,and liver in all the drainage groups were respectively lower than the corresponding non-drainage groups [jejunum:PUFA (I/R) vs EN (I/R) vs N (I/R) vs PUFA (I/R+D) vs EN (I/R+D) vsN (I/R+D):2.32±0.62vs3.08±1.29vs3.50±2.44vs 1.62±0.79vs 1.67±1.11 vs 1.94±0.81,P=0.025; ileum:PUFA (1/R) vsEN (1/R) vsN (1/R) vs PUFA (1/R+D) vsEN (1/R+D) vs N (1/R+D):2.67±1.08 vs 5.22 ± 3.96 vs 6.95 ±4.92 vs 1.70±0.68 vs 1.80±0.29 vs3.68±1.47,P=0.012; liver:PUFA (1/R)vsEN (1/R)vsN (1/R)vs PUFA (1/R+D)vsEN (1/R+D)vsN (1/R+D):5.67 ±1.94 vs 7.50 ±3.89 vs 7.18 ±4.55 vs 1.70 ±0.86 vs 3.90 ± 1.95 vs 4.12 ±2.11,P =0.001],which was consistent with the reduction of HMGB1 and the decrease of nuclear factor-κB activity in intestine,liver,and lung (P =0.000).Conclusions Lymph drainage and ω-3 PUFAs intervention can reduce the production of HMGB1 and inflammation factors,inhibit the expression of HMGB1 and TLR4 mRNA,and thus alleviate distant tissue injury caused by intestinal L/R.